The present invention is concerned with the use of a derivatized alkaline phosphatase as standard for the determination of human alkaline phosphatase.
Alkaline phosphatase (EC 3.1.3.1) (AP), an enzyme which hydrolyzes the esters of phosphoric acid, is essentially formed in the body in the liver, in bones, in the kidneys, in the small intestine and, in the case of pregnant females, also in the placenta. The individual isoenzymes differ, in particular, by their carbohydrate content. In the case of diseases of the organs, in each case the isoenzyme formed in these organs passes in increased amounts into the blood circulation. Therefore, the detection of the individual enzymes in the serum is diagnostically very valuable.
As a rule, the proportion of kidney, small intestine and placenta phosphatase in the serum is small. Furthermore, small intestine and placenta phosphatase can be very easily separated from the other isoenzymes by conventional separation processes, such as chromatography. Of especial interest is the differentiation of alkaline phosphatase formed in the bones or in the liver. In this manner, there are obtained indications of carcinomas, bone disease and the type and stage of liver diseases. Therefore, in the scope of clinical diagnosis, it is important to determine not only the total concentration of alkaline phosphatase in the blood but also the proportion of the two isoenzymes.
One possibility for this purpose is provided by the different behaviour of the two isoenzymes with regard to wheat germ lectin. Whereas the alkaline bone phosphatase reacts immediately with wheat germ lectin and precipitates out, the liver phosphatase remains longer in solution and only slowly forms insoluble complexes with wheat germ lectin.
In Arztl. Lab., 32, 159-165/1986, there has been described a process for the quantitative determination of alkaline total phosphatase, as well as of alkaline bone phosphatase, in which the total content of alkaline phosphatase is first determined in a sample. Subsequently, the alkaline bone phosphatase is precipitated out with wheat germ lectin and the amount of alkaline liver phosphatase is determined in the supernatant. From these two values there can then be determined, in a simple manner, the amount of alkaline bone phosphatase.
A problem in the case of this method of determination is that no standard solutions are available. Since the determination of the amount of the alkaline bone phosphatase takes place after a precipitation reaction, for the exactitude of the determination it would be important to determine in a comparison solution, the content of the individual isoenzymes of which is known, which amount of the isoenzymes is to be found in which fraction. Therefore, for precise determinations, the provision of a standard solution is desirable.
In the case of the previously commercially available tests, for the control there is only used a phosphatase which cannot be precipitated with wheat germ lectin. This is disadvantageous since it is precisely the precipitating out of the phosphatases which contains the possibilities of error which are to be determined by testing with a standard solution. A further problem is that for the standard solution a phosphatase must be made available, the activity of which corresponds to that of the phosphatase to be detected in the blood.
Since the isoenzymes extracted from the organs are not identical with the corresponding isoenzymes in the serum and, therefore, display a different precipitation behaviour with regard to wheat germ lectin and since for the enrichment from serum insufficiently large amounts are available, it is an object of the present invention to provide a standard solution for use in the differentiated determination of alkaline phosphatase.